A comprehensive SARS-CoV-2 and COVID-19 review, Part 2: host extracellular to systemic effects of SARS-CoV-2 infection.

COVID-19, the disease caused by SARS-CoV-2, has caused significant morbidity and mortality worldwide. The betacoronavirus continues to evolve with global health implications as we race to learn more to curb its transmission, evolution, and sequelae. The focus of this review, the second of a three-part series, is on the biological effects of the SARS-CoV-2 virus on post-acute disease in the context of tissue and organ adaptations and damage. We highlight the current knowledge and describe how virological, animal, and clinical studies have shed light on the mechanisms driving the varied clinical diagnoses and observations of COVID-19 patients. Moreover, we describe how investigations into SARS-CoV-2 effects have informed the understanding of viral pathogenesis and provide innovative pathways for future research on the mechanisms of viral diseases.

COVID-19 and candiduria: an investigation of the risk factors and immunological aspects.

Secondary fungal infections are frequently observed in COVID-19 patients. However, the occurrence of candiduria in these patients and its risk factors are underexplored. We evaluated the risk factors of candiduria in COVID-19 patients, including inflammatory mediators that could be used as prognostic markers. Clinical information, laboratory test results, and outcomes were collected from severely ill COVID-19 patients with and without candiduria. Candida species identification, antifungal susceptibility, and plasma inflammatory mediators' measurements were performed. Regression logistic and Cox regression model were used to evaluate the risk factors. A higher risk of longer hospitalization and mortality were observed in patients with candiduria compared to those with COVID-19 only. Candiduria was caused by Candida albicans, C. glabrata, and C. tropicalis. Isolates with intermediate susceptibility to voriconazole and resistant to caspofungin were identified. Classic factors such as the use of corticosteroids and antibacterials, the worsening of renal function, and hematological parameters (hemoglobin and platelets) were found to predispose to candiduria. The mediators IL-1ß, IL-1ra, IL-2, CXCL-8, IL-17, IFN-γ, basic FGF, and MIP-1ß were significantly increased in patients with COVID-19 and candiduria. Furthermore, IFN-γ, IL-1ra, and CXCL-8 were associated with the occurrence of candiduria in COVID-19 patients, whereas basic FGF, IL-1ß, and CXCL-8 were associated with the risk of death in these patients. Classical and immunological factors were associated with worse prognosis among patients with COVID-19 and candiduria. Some mediators, especially CXCL-8, can be a reliable biomarker of fungal coinfection and may guide the diagnostic and the treatment of these patients.

New, fast, and precise method of COVID-19 detection in nasopharyngeal and tracheal aspirate samples combining optical spectroscopy and machine learning.

Fast, precise, and low-cost diagnostic testing to identify persons infected with SARS-CoV-2 virus is pivotal to control the global pandemic of COVID-19 that began in late 2019. The gold standard method of diagnostic recommended is the RT-qPCR test. However, this method is not universally available, and is time-consuming and requires specialized personnel, as well as sophisticated laboratories. Currently, machine learning is a useful predictive tool for biomedical applications, being able to classify data from diverse nature. Relying on the artificial intelligence learning process, spectroscopic data from nasopharyngeal swab and tracheal aspirate samples can be used to leverage characteristic patterns and nuances in healthy and infected body fluids, which allows to identify infection regardless of symptoms or any other clinical or laboratorial tests. Hence, when new measurements are performed on samples of unknown status and the corresponding data is submitted to such an algorithm, it will be possible to predict whether the source individual is infected or not. This work presents a new methodology for rapid and precise label-free diagnosing of SARS-CoV-2 infection in clinical samples, which combines spectroscopic data acquisition and analysis via artificial intelligence algorithms. Our results show an accuracy of 85% for detection of SARS-CoV-2 in nasopharyngeal swab samples collected from asymptomatic patients or with mild symptoms, as well as an accuracy of 97% in tracheal aspirate samples collected from critically ill COVID-19 patients under mechanical ventilation. Moreover, the acquisition and processing of the information is fast, simple, and cheaper than traditional approaches, suggesting this methodology as a promising tool for biomedical diagnosis vis-à-vis the emerging and re-emerging viral SARS-CoV-2 variant threats in the future.

Multi-Epitope Protein as a Tool of Serological Diagnostic Development for HTLV-1 and HTLV-2 Infections.

A multi-epitope protein expressed in a prokaryotic system, including epitopes of Env, Gag, and Tax proteins of both HTLV-1 and HTLV-2 was characterized for HTLV-1/2 serological screening. This tool can contribute to support the implementation of public policies to reduce HTLV-1/2 transmission in Brazil, the country with the highest absolute numbers of HTLV-1/2 infected individuals. The chimeric protein was tested in EIA using serum/plasma of HTLV-infected individuals and non-infected ones from four Brazilian states, including the North and Northeast regions (that present high prevalence of HTLV-1/2) and Southeast region (that presents intermediate prevalence rates) depicting different epidemiological context of HTLV-1/2 infection in our country. We enrolled samples from Pará (n = 114), Maranhão (n = 153), Minas Gerais (n = 225) and São Paulo (n = 59) states; they are from blood donors' candidates (Pará and Minas Gerais), pregnant women (Maranhão) and HIV+/high risk for sexually transmitted infection (STI; São Paulo). Among the HTLV-1/2 positive sera, there were co-infections with viral (HTLV-1 + HTLV-2, HIV, HCV, and HBV), bacterial (Treponema pallidum) and parasitic (Trypanosoma cruzi, Schistosma mansoni, Strongyloides stercoralis, Entamoeba coli, E. histolytica, and Endolimax nana) pathogens related to HTLV-1/2 co-morbidities that can contribute to inconclusive diagnostic results. Sera positive for HIV were included among the HTLV-1/2 negative samples. Considering both HTLV-1 and HTLV-2-infected samples from all states and different groups (blood donor candidates, pregnant women, and individuals with high risk for STI), mono or co-infected and HTLV-/HIV+, the test specificity ranged from 90.09 to 95.19% and the sensitivity from 82.41 to 92.36% with high accuracy (ROC AUC = 0.9552). This multi-epitope protein showed great potential to be used in serological screening of HTLV-1 and HTLV-2 in different platforms, even taking into account the great regional variation and different profile of HTLV-1 and HTLV-2 mono or co-infected individuals.

Multi-Epitope Protein as a Tool of Serological Diagnostic Development for HTLV-1 and HTLV-2 Infections

A multi-epitope protein expressed in a prokaryotic system, including epitopes of Env, Gag, and Tax proteins of both HTLV-1 and HTLV-2 was characterized for HTLV-1/2 serological screening. This tool can contribute to support the implementation of public policies to reduce HTLV-1/2 transmission in Brazil, the country with the highest absolute numbers of HTLV-1/2 infected individuals. The chimeric protein was tested in EIA using serum/plasma of HTLV-infected individuals and non-infected ones from four Brazilian states, including the North and Northeast regions (that present high prevalence of HTLV-1/2) and Southeast region (that presents intermediate prevalence rates) depicting different epidemiological context of HTLV-1/2 infection in our country. We enrolled samples from Pará (n = 114), Maranhão (n = 153), Minas Gerais (n = 225) and São Paulo (n = 59) states; they are from blood donors' candidates (Pará and Minas Gerais), pregnant women (Maranhão) and HIV+/high risk for sexually transmitted infection (STI; São Paulo). Among the HTLV-1/2 positive sera, there were co-infections with viral (HTLV-1 + HTLV-2, HIV, HCV, and HBV), bacterial (Treponema pallidum) and parasitic (Trypanosoma cruzi, Schistosma mansoni, Strongyloides stercoralis, Entamoeba coli, E. histolytica, and Endolimax nana) pathogens related to HTLV-1/2 co-morbidities that can contribute to inconclusive diagnostic results. Sera positive for HIV were included among the HTLV-1/2 negative samples. Considering both HTLV-1 and HTLV-2-infected samples from all states and different groups (blood donor candidates, pregnant women, and individuals with high risk for STI), mono or co-infected and HTLV-/HIV+, the test specificity ranged from 90.09 to 95.19% and the sensitivity from 82.41 to 92.36% with high accuracy (ROC AUC = 0.9552). Thismulti-epitope protein showed great potential to be used in serological screening of HTLV-1 and HTLV-2 in different platforms, even taking into account the great regional variation and different profile of HTLV-1 and HTLV-2 mono or co-infected individuals. (AU)

Will a little change do you good? A putative role of polymorphisms in COVID-19.

An alarming disease caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) named COVID-19 has emerged as an unprecedented public health problem and ignited a world health crisis. As opposed to what was believed at the beginning of the pandemic, the virus has not only spread but persevere causing secondary waves and challenging the concept of herd immunity against viral infections. While the majority of SARS-CoV-2-infected individuals may remain asymptomatic, a fraction of individuals may develop low to high-grade severity signs and symptoms of COVID-19. The disease is multifactorial and can progress quickly, leading to severe complications and even death in a few days. Therefore, understanding the pre-existing factors for disease development has never been so pressing. In this scenario, the insights on the mechanisms underlying disease allied to the immune response developed during the viral invasion could shed light on novel predictive factors and prognostic tools for COVID-19 management and interventions. A recent genome-wide association study (GWAS) revealed several molecules that significantly impacted critically ill COVID-19 patients, leading to the core mechanisms of COVID-19 pathogenesis. Considering these findings and the fact that ACE-2 polymorphisms alone cannot explain disease progress and severity, this review aims at summarizing the most important and recent findings of the research and expert consensus of possible cytokine-related polymorphisms existing in the differential expression of paramount immune molecules that could be crucial for providing guidelines for decision-making and appropriate clinical management of COVID-19.

A chimeric HLA-A2:ß2M:Ig fusion protein for the study of virus-specific CD8+ T-cells.

INTRODUCTION: The response mediated by CD8+ T-cells in the context of infection and vaccination has been thoroughly investigated and represents one of the most important branches that allow for the development of immunity against intracellular pathogens and, thus, the establishment of robust antiviral responses. However, there is a lack of methods to assess antigen-specific CD8+ T-cells. OBJECTIVE: Search for the ideal assays to assess the function of antigen-specific CD8+ T-cells. METHODS: In the present study a chimeric HLA-A2:ß2M:Ig fusion protein was produced, purified, and evaluated in functional CD8+ T-cell response studies using samples from Influenza A patients and humanized mice upon adenoviral vaccination. RESULTS: The HLA-A2:ß2M:Ig molecule, bound to immunodominant viral peptides by passive transfer, was able to induce robust antiviral CD8+ T-cell responses mediated by IFN-γ. The in vitro IFN-γ release assay using the chimeric HLA-A2:ß2M:Ig fusion protein detected bona fide human CD8+ T-cells, demonstrating superior production of IFN-γ by human CD8+ T-cells induced by Influenza A immunodominant GILGFVFTL peptide. Removal of antigen-presenting cells and CD8+ T-cell enrichment improved significantly the IFN-γ production. The chimeric HLA-A2:ß2M:Ig fusion protein also triggered HLA-A2-restricted CD8+ T-cell response in a humanized mouse model upon vaccination with adenovirus encoding HLA-A2-restricted HIV p24 antigen. The results strongly suggest the use of tailor-made assays for detecting HLA-A2-restricted CD8+ T-cell Responses in the Humanized Mouse Model. CONCLUSION: The chimeric HLA-A2:ß2M:Ig fusion protein-based assays provided a sensitive tool that may be paramount to measure virus-specific CD8+ T-cell response in a range of viral infections of clinical relevance.

Functional Human CD141+ Dendritic Cells in Human Immune System Mice.

BACKGROUND: For the purpose of studying functional human dendritic cells (DCs) in a humanized mouse model that mimics the human immune system (HIS), a model referred to as HIS mice was established. METHODS: Human immune system mice were made by engrafting NOD/SCID/IL2Rgammanull (NSG) mice with human hematopoietic stem cells (HSCs) following the transduction of genes encoding human cytokines and human leukocyte antigen (HLA)-A2.1 by adeno-associated virus serotype 9 (AAV9) vectors. RESULTS: Our results indicate that human DC subsets, such as CD141+CD11c+ and CD1c+CD11c+ myeloid DCs, distribute throughout several organs in HIS mice including blood, bone marrow, spleen, and draining lymph nodes. The CD141+CD11c+ and CD1c+CD11c+ human DCs isolated from HIS mice immunized with adenoviruses expressing malaria/human immunodeficiency virus (HIV) epitopes were able to induce the proliferation of malaria/HIV epitopes-specific human CD8+ T cells in vitro. Upregulation of CD1c was also observed in human CD141+ DCs 1 day after immunization with the adenovirus-based vaccines. CONCLUSIONS: Establishment of such a humanized mouse model that mounts functional human DCs enables preclinical assessment of the immunogenicity of human vaccines in vivo.

Cytokines as biomarkers to monitoring the impact of multidrug therapy in immune response of leprosy patients.

Leprosy or Hansen's disease is a chronic infectious disease of the skin and nerves, caused by the intracellular bacilli Mycobacterium leprae. It is characterized by a spectrum of clinical forms depending on the host's immune response to M. leprae. Patients with tuberculoid (TT) leprosy have strong cell-mediated immunity (CMI) with elimination of the bacilli, whereas patients with lepromatous (LL) leprosy exhibit defective CMI to M. leprae. Despite advances in the understanding of the pathogenesis of leprosy and the development of new therapeutic strategies, there is a need for the identification of biomarkers which be used for early diagnosis and to discrimination between different forms of the disease, as prognostic markers. Here, we analyzed the serum levels of IL-1ß, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFN-γ and TNF in order to address the contribution of these cytokines in late phase of M. leprae infection, and the impact of multidrug therapy (MDT). Our results demonstrated that patients of LL group presented higher expression of serum levels of inflammatory cytokines before MDT, while TT patients presented a balance between inflammatory and regulatory cytokines. MDT changes the profile of serum cytokines in M. leprae infected patients, as evidenced by the cytokine network, especially in TT patients. LL patients displayed a multifaceted cytokine system characterized by strong connecting axes involving inflammatory/regulatory molecules, while TT patients showed low involvement of regulatory cytokines in network overall. Cytokines can be identified as good biomarkers of the impact of MDT on the immune system and the effectiveness of treatment.

Distinct patterns of cellular immune response elicited by influenza non-adjuvanted and AS03-adjuvanted monovalent H1N1(pdm09) vaccine.

The study aimed at identifying biomarkers of immune response elicited by non-adjuvanted-(NAV) and adjuvanted-(AV) H1N1(pdm09) vaccines. The results showed that despite both vaccines elicited similar levels of anti-H1N1 antibodies at day30 after vaccination, higher reactivity was observed in AV at day180. While AV induced early changes in cell-surface molecules on monocytes, CD4+, CD8+ T-cells and B-cells, NAV triggered minor changes, starting later on at day3. Furthermore, AV induced a late and persistent increase in TLR gene expression after day3, except for tlr4, while NAV displayed earlier but transient tlr3/4/7/9 up-regulation. Contrasting with NAV, prominent chemokine gene expression (cxcl8,cxcl9,ccl5) and a broad spectrum up-regulation of plasmatic biomarkers (CXCL8,IL-6,IL-1ß,IL-12,IL-10) was evident in AV, which showed a major involvement of TNF and IL-10. Similarly, AV induced a robust IL-10-modulated proinflammatory storm, with early and persistent involvement of TNF-α/IL-12/IFN-γ axis derived from NK-cells, CD4+ and CD8+ T-cells along with promiscuous production of IL-4/IL-5/IL-13. Conversely, NAV promotes a concise and restricted intracytoplasmic chemokine/cytokine response, essentially mediated by TNF-α and IL-4, with late IL-10 production by CD8+ T-cells. Systems biology approach underscored that AV guided the formation of an imbricate network characterized by a progressive increase in the number of neighborhood connections amongst innate and adaptive immunity. In AV, the early cross-talk between innate and adaptive immunity, followed by the triad NK/CD4+/CD8+ T-cells at day3, sponsored a later/robust biomarker network. These findings indicate the relevance of adjuvanted vaccination to orchestrate broad, balanced and multifactorial cellular immune events that lead ultimately to a stronger H1N1 humoral immunity.

Co-administration of α-GalCer analog and TLR4 agonist induces robust CD8(+) T-cell responses to PyCS protein and WT-1 antigen and activates memory-like effector NKT cells.

In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8(+) T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8(+) T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii, and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immunization regimens were able to induce higher levels of CD8(+) T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44(+)CD62L(-)NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation.

Booster dose after 10 years is recommended following 17DD-YF primary vaccination.

A single vaccination of Yellow Fever vaccines is believed to confer life-long protection. In this study, results of vaccinees who received a single dose of 17DD-YF immunization followed over 10 y challenge this premise. YF-neutralizing antibodies, subsets of memory T and B cells as well as cytokine-producing lymphocytes were evaluated in groups of adults before (NVday0) and after (PVday30-45, PVyear1-4, PVyear5-9, PVyear10-11, PVyear12-13) 17DD-YF primary vaccination. YF-neutralizing antibodies decrease significantly from PVyear1-4 to PVyear12-13 as compared to PVday30-45, and the seropositivity rates (PRNT≥2.9Log10mIU/mL) become critical (lower than 90%) beyond PVyear5-9. YF-specific memory phenotypes (effector T-cells and classical B-cells) significantly increase at PVday30-45 as compared to naïve baseline. Moreover, these phenotypes tend to decrease at PVyear10-11 as compared to PVday30-45. Decreasing levels of TNF-α(+) and IFN-γ(+) produced by CD4(+) and CD8(+) T-cells along with increasing levels of IL-10(+)CD4(+)T-cells were characteristic of anti-YF response over time. Systems biology profiling represented by hierarchic networks revealed that while the naïve baseline is characterized by independent micro-nets, primary vaccinees displayed an imbricate network with essential role of central and effector CD8(+) memory T-cell responses. Any putative limitations of this cross-sectional study will certainly be answered by the ongoing longitudinal population-based investigation. Overall, our data support the current Brazilian national immunization policy guidelines that recommend one booster dose 10 y after primary 17DD-YF vaccination.

Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria.

In this study, we developed human immune system (HIS) mice that possess functional human CD4+ T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4+ T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4+ T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4+ T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4+ T cell responses both specific for a human malaria antigen.

Previous Ingestion of Lactococcus lactis by Ethanol-Treated Mice Preserves Antigen Presentation Hierarchy in the Gut and Oral Tolerance Susceptibility.

BACKGROUND: Ethanol (EtOH) consumption is able to disturb the ovalbumin (OVA)-oral tolerance induction by interfering on the function of antigen presenting cells (APC), down-regulating dendritic cells (DCs) and macrophages and up-regulating B-lymphocytes and their function, which results in an overall allergic-type immune status. In this study, the potential of a priori administration of Lactococcus lactis (LL) in avoiding loss of oral tolerance in EtOH-treated mice was investigated. METHODS: Female C57BL/6 mice received, by oral route, ad libitum wild-type (WT) LL or heat-shock protein producer (Hsp65) LL for 4 consecutive days. Seven days later, mice were submitted to short-term high-dose EtOH treatment. After 24 hours, stomach, intestine, spleen, mesenteric lymph nodes (mLN) specimens were collected for biomarkers analysis. Following EtOH-treatment protocol, a group of animals underwent single-gavage OVA-tolerance protocol and sera samples collected for antibody analysis. RESULTS: The ingestion of WT LL or Hsp65 LL is able to restore oral tolerance to OVA in EtOH-treated mice, by reducing local and systemic allergic outcomes such as gastric mast cells and gut-interleukin-4, as well as serum IgE. WT LL treatment prevents the decrease of mLN regulatory T cells induced by the EtOH treatment. Moreover, LL treatment preserves APC hierarchy and antigen presentation commitment in EtOH-treated mice, with conserved DC and macrophage activity over B lymphocytes in mLN and preserved macrophage activity over DC and B-cell subsets in the spleen. CONCLUSIONS: The present findings suggest that a priori ingestion of LL preserves essential mechanisms associated with oral tolerance induction that are disturbed by EtOH ingestion. Maintenance of mucosal homeostasis by preserving APC hierarchy and antigen presentation commitment could be associated with T-regulatory subset activities in the gastrointestinal tract.

CD11c+CD123Low dendritic cell subset and the triad TNF-α/IL-17A/IFN-γ integrate mucosal and peripheral cellular responses in HIV patients with high-grade anal intraepithelial neoplasia: a systems biology approach.

BACKGROUND: The incidence of anal cancer has increased over the past 25 years, and HIV/HPV coinfection is the most important risk factor for anal squamous cell carcinoma. In this study, we demonstrated that the evaluation of systemic and compartmentalized anal mucosa immune response is relevant to differentiating HIV(+) patients at risk of anal intraepithelial neoplasia (AIN). METHODS: A systems biology approach was used to integrate different immunological parameters from anal mucosal tissue and peripheral blood assessed by phenotypic and intracytoplasmic analysis of lymphocytes and dendritic cell subsets. RESULTS: Our data demonstrated that anal mucosal mononuclear cells from AIN(+)HIV(+) patients showed a robust capacity in producing proinflammatory/regulatory cytokines, mainly mTNF-α > IL-4 > IL-10 > IL-6 = IL-17A. Mucosal TNF-α/IFN-γ/IL-17A are selective high-grade squamous intraepithelial lesion (HSIL)-related biomarkers. Higher levels of circulating CD11cCD123cells and CD1a cells along with elevated levels of IFN-γCD4 T cells are major features associated with HSIL in AIN(+)HIV(+) patients. Regardless of the presence of AIN, HIV(+) patients presented a complex biomarker network, rich in negative connections. Among those patients, however, HSIL+ patients displayed stronger positive links between peripheral blood and anal mucosa environments, exemplified by the subnet of IL-17A/TNF-α/CD4IFN-γ/CD11cCD123 cells. CONCLUSIONS: The significant association between HSIL and the levels of TNF-α/IL-17A/IFN-γ along with the different subsets of DCs present in the anal mucosa milieu should be studied in more detail as a way to identify and categorize HIV(+) patients vis à vis the high risk of anal cancer outcome.

Biomarker analysis revealed distinct profiles of innate and adaptive immunity in infants with ocular lesions of congenital toxoplasmosis.

Toxoplasma gondii is the main infectious cause of human posterior retinochoroiditis, the most frequent clinical manifestation of congenital toxoplasmosis. This investigation was performed after neonatal screening to identify biomarkers of immunity associated with immunopathological features of the disease by flow cytometry. The study included infected infants without NRL and with retinochoroidal lesions (ARL, ACRL, and CRL) as well as noninfected individuals (NI). Our data demonstrated that leukocytosis, with increased monocytes and lymphocytes, was a relevant hematological biomarker of ARL. Immunophenotypic analysis also revealed expansion of CD14(+)CD16(+)HLA-DR(high) monocytes and CD56(dim) cytotoxic NK-cells in ARL. Moreover, augmented TCRγ δ (+) and CD8(+) T-cell counts were apparently good biomarkers of morbidity. Biomarker network analysis revealed that complex and intricated networks underscored the negative correlation of monocytes with NK- and B-cells in NRL. The remarkable lack of connections involving B-cells and a relevant shift of NK-cell connections from B-cells toward T-cells observed in ARL were outstanding. A tightly connected biomarker network was observed in CRL, with relevant connections of NK- and CD8(+) T-cells with a broad range of cell subsets. Our findings add novel elements to the current knowledge on the innate and adaptive immune responses in congenital toxoplasmosis.

A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.

An AAV vector-mediated gene delivery approach facilitates reconstitution of functional human CD8+ T cells in mice.

In the present study, a novel adeno-associated virus (AAV) vector-mediated gene delivery approach was taken to improve the reconstitution of functional CD8(+) T cells in humanized mice, thereby mimicking the human immune system (HIS). Human genes encoding HLA-A2 and selected human cytokines (A2/hucytokines) were introduced to an immune-deficient mouse model [NOD/SCID/IL2rγ(null) (NSG) mice] using AAV serotype 9 (AAV9) vectors, followed by transplantation of human hematopoietic stem cells. NSG mice transduced with AAV9 encoding A2/hucytokines resulted in higher levels of reconstitution of human CD45(+) cells compared to NSG mice transduced with AAV9 encoding HLA-A2 alone or HLA-A2-transgenic NSG mice. Furthermore, this group of HIS mice also mounted the highest level of antigen-specific A2-restricted human CD8(+) T-cell response upon vaccination with recombinant adenoviruses expressing human malaria and HIV antigens. Finally, the human CD8(+) T-cell response induced in human malaria vaccine-immunized HIS mice was shown to be functional by displaying cytotoxic activity against hepatocytes that express the human malaria antigen in the context of A2 molecules. Taken together, our data show that AAV vector-mediated gene delivery is a simple and efficient method to transfer multiple human genes to immune-deficient mice, thus facilitating successful reconstitution of HIS in mice. The HIS mice generated in this study should ultimately allow us to swiftly evaluate the T-cell immunogenicity of various human vaccine candidates in a pre-clinical setting.

A glycolipid adjuvant, 7DW8-5, enhances CD8+ T cell responses induced by an adenovirus-vectored malaria vaccine in non-human primates.

A key strategy to a successful vaccine against malaria is to identify and develop new adjuvants that can enhance T-cell responses and improve protective immunity. Upon co-administration with a rodent malaria vaccine in mice, 7DW8-5, a recently identified novel analog of α-galactosylceramide (α-GalCer), enhances the level of malaria-specific protective immune responses more strongly than the parent compound. In this study, we sought to determine whether 7DW8-5 could provide a similar potent adjuvant effect on a candidate human malaria vaccine in the more relevant non-human primate (NHP) model, prior to committing to clinical development. The candidate human malaria vaccine, AdPfCA (NMRC-M3V-Ad-PfCA), consists of two non-replicating recombinant adenoviral (Ad) vectors, one expressing the circumsporozoite protein (CSP) and another expressing the apical membrane antigen-1 (AMA1) of Plasmodium falciparum. In several phase 1 clinical trials, AdPfCA was well tolerated and demonstrated immunogenicity for both humoral and cell-mediated responses. In the study described herein, 25 rhesus macaques received prime and boost intramuscular (IM) immunizations of AdPfCA alone or with an ascending dose of 7DW8-5. Our results indicate that 7DW8-5 is safe and well-tolerated and provides a significant enhancement (up to 9-fold) in malaria-specific CD8+ T-cell responses after both priming and boosting phases, supporting further clinical development.

Effects of HIV-1-induced CD1c and CD1d modulation and endogenous lipid presentation on CD1c-restricted T-cell activation.

BACKGROUND: It has been shown that human immunodeficiency virus (HIV)-1 infection induces the production of endogenous lipids required for effective viral production, and the cluster of differentiation (CD)1 molecule CD1d is downregulated by HIV-1 infection. However, the role of endogenous lipid presentation and the implications of CD1 downregulation by HIV-1 infection have not yet been characterized. RESULTS: In this study, we observed downregulation of both CD1c and CD1d expression through a Vpu-dependent and Nef-independent mechanism, and the concomitant HIV-1-induced production of host cholesterol decreased the extent of CD1c and CD1d modulation. While the modest downregulation of CD1c by HIV-1 infection decreased the ability of CD1c-restricted T cells to respond and secrete interferon-γ, the cholesterol upregulation in the same cells by HIV-1 infection appears to limit the downregulation of CD1c. CONCLUSIONS: The two conflicting HIV-1-mediated changes in CD1c expression appear to minimize the modulation of CD1c expression, thus leading the host to maintain a CD1c-restricted T-cell response against HIV-1.